Stability and oligosaccharide binding of the N1 cellulose-binding domain of Cellulomonas fimi endoglucanase CenC.

Abstract

Differential scanning calorimetry has been used to study the thermal stability and oligosaccharide-binding thermodynamics of the N-terminal cellulose-binding domain of Cellulomonas fimi beta-1,4-glucanase CenC (CBDN1). CBDN1 has a relatively low maximum stability (delta Gmax = 33 kJ/mol = 216 J/residue at 1 degree C and pH 6.1) compared to other small single-domain globular proteins. The unfolding is fully reversible between pH 5.5 and 9 and in accordance with the two-state equilibrium model between pH 5.5 and 11. When the single disulfide bond in CBDN1 is reduced, the protein remains unfolded at all conditions, as judged by NMR spectroscopy. This indicates that the intramolecular cross-link makes a major contribution to the stability of CBDN1. The measured heat capacity change of unfolding (delta Cp = 7.5 kJ mol-1 K-1) agrees well with that calculated from the predicted changes in the solvent accessible nonpolar and polar surface areas upon unfolding. Extrapolation of the specific enthalpy and entropy of unfolding to their respective convergence temperature indicates that per residue unfolding energies for CBDN1, an isolated domain, are in accordance with those found by Privalov (1) for many single-domain globular proteins. DSC thermograms of the unfolding of CBDN1 in the presence of various concentrations of cellopentaose were fit to a thermodynamic model describing the linkage between protein-ligand binding and protein unfolding. A global two-dimensional minimization routine is used to regress the binding enthalpy, binding constant, and unfolding thermodynamics for the CBDN1-cellopentaose system. Extrapolated binding constants are in quantitative agreement with those determined by isothermal titration calorimetry at 35 degrees C. Study holds ProTherm entries: 17827, 17828, 17829, 17830, 17831, 17832, 17833, 17834, 17835, 17836, 17837, 17838, 17839, 17840, 17841, 17842, 17843, 17844 Extra Details: cellulose binding domain (CBD), endonuclease CenC, cellulomonas fimi, cellopentaose.

Submission Details

ID: RUJcJy7H4

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:50 p.m.

Version: 1

Publication Details
Creagh AL;Koska J;Johnson PE;Tomme P;Joshi MD;McIntosh LP;Kilburn DG;Haynes CA,Biochemistry (1998) Stability and oligosaccharide binding of the N1 cellulose-binding domain of Cellulomonas fimi endoglucanase CenC. PMID:9521674
Additional Information

Study Summary

Number of data points 28
Proteins Endoglucanase C ; Endoglucanase C
Unique complexes 1
Assays/Quantities/Protocols Experimental Assay: dG temp:298 K, buffers:dibasic sodium phosphate, sodium hydroxide: 50 mM, pH:11.06 ; Experimental Assay: dG temp:298 K, buffers:glycine, sodium hydroxide: 50 mM, pH:10.86 ; Experimental Assay: dG temp:298 K, pH:10.58, buffers:glycine, sodium hydroxide: 50 mM ; Experimental Assay: dG pH:9.06, temp:298 K, buffers:glycine, sodium hydroxide: 50 mM ; Experimental Assay: dG temp:298 K, buffers:dibasic and monobasic sodium phosphate: 50 mM, pH:7.36 ; Experimental Assay: dG temp:298 K, pH:7.09, buffers:dibasic and monobasic sodium phosphate: 50 mM ; Experimental Assay: dG temp:298 K, buffers:dibasic and monobasic sodium phosphate: 50 mM, pH:6.1 ; Experimental Assay: dG temp:298 K, buffers:acetic acid, sodium acetate: 50 mM, pH:5.5 ; Experimental Assay: dG buffers:acetic acid, sodium acetate: 50 mM, pH:5.14 ; Experimental Assay: dHcal buffers:dibasic sodium phosphate, sodium hydroxide: 50 mM, pH:11.06 ; Experimental Assay: Tm buffers:dibasic sodium phosphate, sodium hydroxide: 50 mM, pH:11.06 ; Experimental Assay: dHcal buffers:glycine, sodium hydroxide: 50 mM, pH:10.86 ; Experimental Assay: Tm buffers:glycine, sodium hydroxide: 50 mM, pH:10.86 ; Experimental Assay: dHcal pH:10.58, buffers:glycine, sodium hydroxide: 50 mM ; Experimental Assay: Tm pH:10.58, buffers:glycine, sodium hydroxide: 50 mM ; Experimental Assay: dHcal pH:9.06, buffers:glycine, sodium hydroxide: 50 mM ; Experimental Assay: Tm pH:9.06, buffers:glycine, sodium hydroxide: 50 mM ; Experimental Assay: dHcal buffers:dibasic and monobasic sodium phosphate: 50 mM, pH:7.36 ; Experimental Assay: Tm buffers:dibasic and monobasic sodium phosphate: 50 mM, pH:7.36 ; Experimental Assay: dHcal pH:7.09, buffers:dibasic and monobasic sodium phosphate: 50 mM ; Experimental Assay: Tm pH:7.09, buffers:dibasic and monobasic sodium phosphate: 50 mM ; Experimental Assay: dHcal buffers:dibasic and monobasic sodium phosphate: 50 mM, pH:6.1 ; Experimental Assay: Tm buffers:dibasic and monobasic sodium phosphate: 50 mM, pH:6.1 ; Experimental Assay: dHcal buffers:acetic acid, sodium acetate: 50 mM, pH:5.5 ; Experimental Assay: Tm buffers:acetic acid, sodium acetate: 50 mM, pH:5.5 ; Experimental Assay: dHcal buffers:acetic acid, sodium acetate: 50 mM, pH:5.14 ; Experimental Assay: Tm buffers:acetic acid, sodium acetate: 50 mM, pH:5.14
Libraries Mutations for sequence ASLDSEVELLPHTSFAESLGPWSLYGTSEPVFADGRMCVDLPGGQGNPWDAGLVYNGVPVGEGESYVLSFTASATPDMPVRVLVGEGGGAYRTAFEQGSAPLTGEPATREYAFTSNLTFPPDGDAPGQVAFHLGKAGAYEFCISQVSLTTSAT

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Endoglucanase C P14090 GUNC_CELFA